The aim of this study was to determine microbiological indicators of safety and quality of kulen produced in uncontrolled (n = 9) and controlled conditions (n = 8). The number of lactic acid and enterococci was determined by culture methods and the presence of Listeria monocytogenes by molecular tools (LAMP, Molecular Detection System), antimicrobial susceptibility of isolates was tested and finally sensory analysis of products was performed. The population of lactic acid bacteria and enterococci was higher in kulen samples produced under controlled conditions. Using MALDI-TOF mass spectrometry, the species of Pediococcus pentosaceus, Lactobacillus sakei and Lactobacillus curvatus were determined, which generally are the dominant species in the composition of starter cultures in the production of fermented sausages. In kulen samples made in uncontrolled production conditions, the dominant population of lactic acid bacteria was not isolated, but only the species Staphylococcus epidermidis, Staphylococcus hominis, Staphylococcus warneri and Streptococcus vestibularis were confirmed. Lactic acid bacteria were susceptible to all antibiotics, while staphylococcal strains were most frequently resistant to erythromycin, penicillin, ampicillin, and kanamycin. L. monocytogenes was not determined in any of the kulen samples. Differences in the composition of the dominant microflora may be the reason of product failures and spoilage appearence of kulen from uncontrolled production. The obtained results indicate the need for improvement of traditional production in terms of hygienic and technological standards in order to maintain the indigenous properties of this autochthonous product.